Role of hydrogen sulfide in health and disease

Abstract In the past, hydrogen sulfide (H2S) was recognized as a toxic and dangerous gas; in recent years, with increased research, we have discovered that H2S can act as an endogenous regulatory transmitter. In mammals, H2S‐catalyzing enzymes, such as cystathionine‐β‐synthase, cystathionine‐γ‐lyase, and 3‐mercaptopyruvate sulfurtransferase, are differentially expressed in a variety of tissues and affect a variety of biological functions, such as transcriptional and posttranslational modification of genes, activation of signaling pathways in the cell, and metabolic processes in tissues, by producing H2S. Various preclinical studies have shown that H2S affects physiological and pathological processes in the body. However, a detailed systematic summary of these roles in health and disease is lacking. Therefore, this review provides a thorough overview of the physiological roles of H2S in different systems and the diseases associated with disorders of H2S metabolism, such as ischemia–reperfusion injury, hypertension, neurodegenerative diseases, inflammatory bowel disease, and cancer. Meanwhile, this paper also introduces H2S donors and novel release modes, as well as the latest preclinical experimental results, aiming to provide researchers with new ideas to discover new diagnostic targets and therapeutic options.


INTRODUCTION
Over the years, hydrogen sulfide (H 2 S) has been known for its rotten egg-like odor, toxicity, and environmental hazard.The toxicity of H 2 S is mainly due to the inhibition of cytochrome c oxidase (COX) in mitochondria, leading to chemical asphyxia of cells. 1,2COX is a vital electron transmitter in the respiratory chain, which participates in cellular respiration.Its activity is inhibited, which reduces oxygen utilization in mitochondria, leading to cell hypoxia. 3,4In recent years, human understanding of H 2 S has gradually shifted from toxic substances to gas transmitters with therapeutic drug potential.In 1989, H 2 S was proven to exist in the human brain and may play a certain physiological role. 5In 1996, Japanese scientists demonstrated that H 2 S is a potential signaling molecule that can be produced by cystathionine-β-synthase (CBS) and is involved in neurotransmission. 6The following year, they discovered that cystathionine-γ-lyase (CSE) is another enzyme that produces. 7Subsequently, Wang et al. 8 confirmed that H 2 S is the third physiological signaling molecule, except for carbon monoxide (CO) and nitric oxide (NO).Since then, the field of sulfide research has developed rapidly, and the research results have become richer; in a 2005 paper, Blackstone et al. 9 reported in a pioneering manner that H 2 S can induce a reversible pseudo-death-like state in mice.They hypothesized that H 2 S-mediated induction of pseudo-death may have beneficial medical applications, such as ischemia-reperfusion injury (IRI) or organ preservation after trauma. 97][18][19] For exogenous H 2 S therapy, the manner of H 2 S delivery is important.At present, the main methods of H 2 S delivery include direct inhalation and H 2 S donor.Yet, all these delivery methods have certain limitations.Although recent studies have identified many small molecule H 2 S donors, they still perform poorly regarding stability, solubility, targeting, and half-life.Nowadays, the main way to constitute an H 2 S delivery system is to combine H 2 S donors with polymers by covalent linkage and physical entrapment.The delivery systems optimize the therapeutic efficacy with higher stability and bioavailability compared with small molecule H 2 S donors.
In this article, we will focus on the role of H 2 S in human health and disease.The multiple physiological and pathophysiologic functions of H 2 S will be discussed systemically and comprehensively.We will delve into the novel H 2 S donors in anticipation of expanding new therapeutic ideas for researchers.

General physicochemical properties of H 2 S
H 2 S is a colorless and highly toxic flammable gas with a unique rotten egg or sewage odor.Its molecular weight is 34.08, and its vapor density is heavier than air, making it easier to diffuse at lower points. 10,20As a binary weak acid, hydrosulfuric acid is an aqueous solution of H 2 S that can reach dissociation equilibrium at room temperature (25 • C).The solution concentration in a saturated state is 0.11 mol dm −3 , and its pH value is approximately 4.0.At 37 • C and pH 7.4, pK α1 = 6.76, there is about 20% H 2 S, 80% HS − and a small amount of negligible S 2- in extracellular fluid. 21At the same time, H 2 S is also a small gas molecule with high lipophilicity, which allows it to freely pass through the lipid bilayer structure of the cell membrane. 22H 2 S is a compound similar to water molecules that can be oxidized into sulfur dioxide, sulfate, thiosulfate, and elemental sulfur.In the body, H 2 S can be oxidized to sulfates and thiosulfates, excreted in the urine.Some studies suggest that urinary thiosulfates can serve as one of the biomarkers of H 2 S poisoning. 20,23

Production of endogenous H 2 S
][26] Both CBS and CSE use pyridoxal phosphate (also known as vitamin B6) as cofactors, and their concentrations vary in different tissues. 27,28CBS mainly exists in the central nervous system (CNS) (cerebellum, hippocampus) and liver tissue. 29CSE mainly regulates H 2 S in the cardiovascular system and respiratory system. 15Only present in the cytoplasm, these two enzymes catalyze the conversion of homocysteine to cysteine, generating H 2 S through the reverse sulfur conversion pathway.Research has found that 3-MST is an enzyme involved in endogenous H 2 S production. 30Unlike the two enzymes mentioned earlier, the cofactor of 3-MST is zinc. 20It often cocatalyzes with cysteine aminotransferase (CAT) in mitochondria to produce H 2 S, L-cysteine, and α-Ketoglutaric acid generates F I G U R E 1 Enzyme catalysis of H 2 S production.Endogenous H 2 S can be produced by two ways: enzyme catalysis and nonenzyme catalysis.Enzyme catalysis is the main way and is catalyzed by four enzymes, such as CBS, CSE, 3-MST and DAO.By Figdraw.H 2 S, hydrogen sulfide; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; PLP, pyridoxal-5′-phosphate; 3-MST, 3-mercaptopyruvate sulfurtransferase; 3-MP, 3-methylpyridine; CAT, cysteine aminotransferase; DAO, D-amino acid oxidase.
3-mercaptopyruvate (3-MP) under the catalysis of CAT, and then generates H 2 S and pyruvic acid under the action of 3-MST. 31In 2013, Japanese scientists proposed a new enzyme catalysis pathway. 32This pathway occurs in the peroxisome.4][35] Endogenous H 2 S enzymatic generation pathway (as shown in Figure 1).Some studies have found that when the human body is under oxidative stress or hyperglycemia, the H 2 S produced through nonenzyme catalysis will significantly increase.In red blood cells, the reduction equivalent produced by glucose oxidation can be utilized to reduce elemental sulfur or polysulfides to H 2 S. 20

Metabolism of endogenous H 2 S
In mammals, the excretion of H 2 S varies in different systems.In the respiratory tract, H 2 S is directly excreted as gaseous molecules.Through the urinary tract, H 2 S is mainly excreted as thiosulfate or free sulfate in the urine.However, in the gastrointestinal tract, most H 2 S is still excreted as free sulfide in the feces. 20H 2 S mainly has the following three metabolic pathways: (1) The elimination of H 2 S through the mitochondrial sulfide oxidation pathway, with sulfide quinone oxidoreductase (SQOR) being the key enzyme in this reaction (as shown in Figure 2).First, H 2 S is oxidized to thiosulfate under the catalysis of SQOR. 36,37n this reaction, the primary sulfur acceptor is glutathione (GSH), and the resulting product is glutathione persulfide (GSSH). 38,39In the next step of the reaction, rhodanese (or thiosulfate sulfurtransferase) plays a crucial role as a sulfur transferase that can further oxidize thiosulfate to sulfite or sulfate. 40,41However, due to the rapid oxidation of sulfite to sulfate, H 2 S is ultimately expelled from the body in the form of thiosulfate or sulfate through this pathway. 20,412) Research has found that methylation occurring in the cytoplasm is another metabolic pathway for H 2 S. 26 Thiol-S-methyltransferase (TSMT) can catalyze the conversion of H 2 S to methyl mercaptan and dimethyl sulfide.TSMT is commonly present in cells in the human body but has high activity in mucosal cells of the colon and cecum.42 Compared with the sulfide oxidation pathway, the metabolic process of sulfide methylation is slower.In a study, the rate of sulfide methylation in mammalian colon mucosal cells was approximately 10,000 times slower than the oxidation rate of H 2 S. 43 (3) H 2 S can be cleared by methemoglobin, metal-containing, or sulfur-containing macromolecules.Methemoglobin and myoglobin can promote the binding of H 2 S and iron by regulating the reactivity of iron, accelerating the oxidation rate of H 2 S. 44

H 2 S and cardiovascular regulation
Initially, H 2 S was thought to have a similar role to NO in relaxing blood vessels.As it has been studied more intensively, H 2 S can maintain endothelial cell function by several mechanisms, such as increasing NO bioavailability and stabilizing the translation of endothelial nitric oxide synthase (eNOS). 45,46Early researchers found that H 2 S can cause concentration-dependent vasodilation by opening adenosine triphosphate (ATP)-sensitive potassium (K ATP ) channels in vascular smooth muscle and decreasing extracellular calcium (Ca 2+ ) in flow. 47Later experimental results have shown that glibenclamide can partially inhibit this vasodilation.Nevertheless, the molecular mechanism of how endogenous H 2 S activates K ATP channels remains unclear, and recent studies may fill this gap.It was found that the K ATP protein Kir6.1 undergoes S-Sul hydration when CSE is overexpressed, and another study found that Kir6.1, which undergoes Ssulfation at the Cys43 site, reduces the synthesis of ATP and enhances the function of the K ATP channel, promoting vasodilation. 48,49Meanwhile, H 2 S has been investigated as a possible endothelium-derived relaxing factor, which combines with eNOS to produce S and NO that can activate transient receptor potential (TRP) ankyrin-1 channels, leading to hyperpolarization and vasodilation in endothelial cells and smooth muscle cells. 49Therefore, H 2 S has potential therapeutic possibilities for targeting diseases of the vascular system, but more in-depth mechanistic studies are still needed to determine this.[52] H 2 S also plays a vital role in angiogenesis.Angiogenesis is an essential physiological process in mammals that maintains normal life activities, and it consists of several successive stages, among which the migration of vascular endothelial cells is most important.In earlier studies, researchers found that CSE inhibitors reduced the length of blood vessels in chicken chorioallantoic membrane. 53In another study, exogenous administration of sodium hydrosulfide (NaHS) promoted endothelial cell proliferation and migration, which may be related to the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 54However, these results were not strong evidence that endogenous H 2 S regulates angiogenesis.The researchers then did several more studies, and they found that mice knocked down for CSE had more rapid angiogenesis and wound healing in response to vascular endothelial growth factor (VEGF) stimulation compared with wildtype mice. 53In terms of mechanisms, a growing number of studies have found that both H 2 S and NO play important roles in angiogenesis. 55For example, H 2 S and NO share a common downstream molecule, silent information regulator-1 (SIRT1), the activation of which increases the expression levels of VEGF and cyclic guanosine 3′, 5′-monophosphate (cGMP) and thus participates in the regulation of angiogenesis.In the available reports, the mechanisms by which H 2 S and NO promote cGMP expression are also different, with H 2 S degrading cGMP by inhibiting phosphodiesterase 5.However, NO promotes soluble guanylyl cyclase production of the cGMP. 56As described above, H 2 S activates Akt phosphorylation and may increase eNOS phosphorylation at the activation site Ser1177. 57Therefore, H 2 S is a physiologically important factor in angiogenesis.

H 2 S and neuromodulation
In an early study in 1989, researchers serendipitously discovered that H 2 S could be detected in the brains of rats even when NaHS was not administered and was prevalent in several regions of the brainstem, cerebellum, hippocampus, and striatum. 5Since then, researchers have studied the interactions between endogenous H 2 S and the nervous system.In 1996, researchers discovered an interesting phenomenon that endogenous H 2 S can enhance N-methyl-daspartate (NMDA) receptors, thus contributing to learning and memory processes. 6They have shown that knockdown of CBS or 3-MST leads to significant impairment of physiological memory formation in mice. 58Another study showed that direct infusion of various CBS inhibitors into the lateral amygdala impaired long-term enhancement of this region, which led to inhibition of cued fear memory formation in rats. 59The concept that H 2 S participates in synaptic neurotransmission has existed since the early 21st century. 60,61Investigators have demonstrated the involvement of CBS-derived H 2 S in nucleus tractus solitarius excitability by using a combination of molecular and pharmacological approaches, providing evidence for the role of endogenous H 2 S in excitatory neurotransmission. 62This may indicate that H 2 S acts presynaptically and involves enhancing intracellular Ca 2+ channels.Overall, most of the studies discussed above were performed in vitro using high (millimolar) NaHS concentrations and could not draw definitive conclusions about the potential physiological relevance of these effects.Therefore, future studies that aim to determine the physiological effects of H 2 S should be conducted using donors in the low micromolar range.

H 2 S and gastrointestinal regulation
In the gastrointestinal tract, under physiological conditions, H 2 S is produced endogenously and exogenously; endogenous is the enzyme origin we mentioned earlier since the two enzymes, CBS and CSE, are present in almost the entire gastrointestinal tract of mammals. 63Regarding the exogenous production of H 2 S, it is mainly the microbiota in the gastrointestinal tract responsible, with sulfate-reducing bacteria being one of the leading producers.Microbial metabolism breaks down proteins in the gastrointestinal tract into amino acids, which include cysteine, H 2 S, and other sulfur-containing compounds. 64,65o, does H 2 S present in the gastrointestinal tract influence gastrointestinal motility?Past studies have given us the answer that the effect of H 2 S on gastrointestinal motility is nonunique.Generally, in vitro studies show that NaHS inhibits smooth muscle contraction in the gastrointestinal tract of different species.7][68][69][70] However, NaHS has a dual effect on spontaneous contractions of gastric smooth muscle in guinea pigs and mice, that is, high concentrations of NaHS inhibit the amplitude of gastric smooth muscle contractions.In contrast, low concentrations increase basal tone. 71,72From the above studies, we can find that the effects of H 2 S on smooth muscle may vary depending on the species and the location of the digestive tract, so the mechanisms also diverge.][75][76]

H 2 S and inflammation
Among the many controversial areas of H 2 S research, the role of H 2 S in inflammatory processes is undoubtedly an example.8][79][80][81][82] For example, the upregulation of CSE induced by lipopolysaccharide (LPS) or proinflammatory cytokines and the consequent increase in H 2 S production can be viewed as a proinflammatory effect or an anti-inflammatory response as a compensatory protective mechanism. 78,83One study found that injection of the H 2 S donor NaHS in rats inhibited leukocyte infiltration and adhesion. 84On the other hand, H 2 S synthesis inhibitors increase leukocyte adhesion, leukocyte infiltration, and edema formation.In animals suffering from acute lung injury caused by burns and smoke inhalation tissue, tissue interleukin (IL)-1 levels were decreased, IL-10 levels were increased in inflamed lungs, and protein oxidation was attenuated after NaHS injection. 82Experimental evidence suggests that H 2 S is a proinflammatory factor in various animal models, including acute pancreatitis, LPS-induced endotoxemia, cecum ligation, and puncture-induced sepsis. 78,85One possible scenario for the proinflammatory mechanism of H 2 S is that H 2 S stimulates sensory nerve endings, releasing endogenous tachykinins such as substance P, calcitonin gene-related peptide, and neurokinin A, leading to neurogenic inflammation.Many factors are involved in determining whether H 2 S is proinflammatory or anti-inflammatory, and the concentration of H 2 S and route of administration may produce different inflammatory outcomes.

H 2 S and aging
The earliest suggestion that the endogenous H 2 S pathway may be regulated during aging was made by researchers Finkelstein and Benevenga, who found that the metabolic capacity of hepatic homogenates to produce volatile sulfur compounds (H 2 S and/or methanethiols) from 3-methylthiopropionate was a product of the metabolism transamination pathway of methionine.All three enzymes, CBS, CSE, and 3-MST, have been reported to show significant age-related downregulation in the brains of aging rats. 868][89][90] A large body of experimental evidence suggests that endogenous or exogenous H 2 S can modulate many core mechanisms implicated in the aging process.These mechanisms include regulation of genome stability, telomere maintenance, epigenetic regu-latory mechanisms, mitochondrial function/dysfunction, stem cell depletion, and protein inhibitory processes.

H 2 S and IRI
According to the progression of diseases, IRI can be divided into two stages: ischemia and reperfusion.It is generally believed that the degree of cell dysfunction, injury, and necrosis is related to the severity and duration of ischemia.Therefore, the main idea for treating IRI is to restore blood flow to the ischemic site as soon as possible. 91However, the sensitivity of different organs to ischemic manifestations also varies, such as the brain, heart, and other organs with poor tolerance to ischemia and hypoxia, and differences in organ tolerance can also affect the degree of cell damage.
In addition, although the reperfusion recovery can provide oxygen and nutrients to cells, it will further strengthen the damage after ischemia, activate cell death and immune response, and so on. 924][95] IRI is a dynamic process with significant organ differences, so a deeper understanding of its molecular mechanisms can help us find better treatment methods.

Ca 2+ overload
When ischemia occurs, ATP in cells is rapidly depleted, ATP synthesis decreases, sodium pump activity decreases, intracellular Na + content increases, and sodium-Ca 2+ exchange proteins are activated, leading to reverse transport of Na + to the extracellular space and an increase in intracellular Ca 2+ . 96,97On the other hand, due to hypoxia and anaerobic metabolism, the production of H + increases, and the pH of extracellular fluid and cytoplasm decreases.When tissue perfusion resumes, the pH of extracellular fluid increases, but the pH of cytoplasm is still very low.In order to reduce the accumulation of H + in cells, H + -Na + exchange protein and Na + -Ca 2+ exchange protein is activated, increasing Ca 2+ overload. 96When the body is in a state of stress, releasing a large amount of catecholamines activates protein kinase C (PKC) through a signaling pathway, promotes H + -Na + exchange, and also increases intracellular Ca 2+ .Due to the massive accumulation of Ca 2+ , the endoplasmic reticulum and mitochondria damage intensifies.The complete opening of the mitochondrial mitochondrial permeability transi- The mechanism of ion exchange leading to Ca 2+ overload during IRI.In IRI, abnormalities in Na + -Ca 2+ exchange are associated with the following three aspects.First, high intracellular Na + directly activates natriuretic proteins during ischemic injury.Second, high intracellular H + decreases pH, which indirectly activates natriuretic proteins.At last, activation of PKC and increased release of catecholamines during reperfusion further promotes Na + -Ca 2+ exchange.By Figdraw.CaBP, calcium binding protein; Ca 2+ , calcium ion; H + , hydrogen ion; Na + , sodium ion; K + , potassium ion.
tion pore (mPTP) will have a more negative impact on cells. 98Figure 3 illustrates the mechanism of Ca 2+ overload during the ischemia and reperfusion phases caused by ion exchange.

ROS
When ischemic tissue undergoes reperfusion, blood brings oxygen and nutrients to the tissue.At the same time, due to the low concentration of antioxidants in cells, the production of reactive oxygen species (ROS) increases.In the I/R process of biology, ROS will be produced in many ways, including mitochondrial electron transfer chain (ETC), xanthine oxidase system (XOD), NADPH oxidase system, nitric oxide synthase (NOS), and so on. 99The first three are related to oxidative stress in multiple organs, such as the heart, brain, lungs, liver, pancreas, kidneys, and gastrointestinal tract. 100NOS mainly acts as an oxidative stress factor in vascular endothelial cells. 101During the metabolism of normal mitochondria, the respiratory chain complex on the inner mitochondrial membrane can produce a small amount of ROS. 102As mentioned earlier, when IRI occurs due to hypoxia, changes in ATP, pH, and Ca 2+ overload occur in cells, which can lead to mitochondrial damage and produce more ROS.However, ROS further exacerbates oxidative stress, leading to a vicious cycle of cells. 96,102he XOD system is an important pathway for ROS production.Under ischemia, ATP synthesis is reduced, and xanthine dehydrogenase is converted into XOD.At the same time, ATP degradation products (ADP, AMP, hypoxanthine) accumulate.When reperfusion is resumed, a large amount of oxygen molecules enters the tissue with the blood.XOD catalyzes the conversion of hypoxanthine into xanthine and uric acid, producing a large amount of ROS. 103The oxygen free radicals generated by this pathway have chemotactic effects, attracting and activating many white blood cells to aggregate.When the tissue resumes its oxygen supply, the activated white blood cells' oxygen consumption increases sharply, producing a large amount of oxygen free radicals, causing cell damage.

Cell death
The IRI process of organisms is dynamic, and the mechanism for producing ROS is also complex.The ROS produced by the above pathways may accumulate in cells during the ischemic stage and inhibit antioxidants.However, after tissue restoration of blood supply, if ischemia is severe, ROS-induced oxidative stress may further cause cell damage and even cell death. 108poptosis is a process of programmed cell death, mainly caused by Ca 2+ overload and ROS activation in IRI.Two cell apoptosis pathways can interact with the endogenous and exogenous apoptosis pathways.The endogenous pathway is also known as the mitochondrial pathway.In the cells injured by ischemia/reperfusion, a large amount of calcium influx and ROS production will cause the opening of mitochondrial mPTP and the activation of apoptosis promoting B-cell lymphoma-2 (Bcl-2) family, increase the permeability of mitochondrial membrane, release cytochrome c into the cytoplasm, and then combine with apoptosis protease activating factor 1 (APAF-1) to activate Caspase-9 and form a complex, and then trigger the apoptosis cascade reaction to promote apoptosis. 109he exogenous pathway, or the death receptor pathway, is mainly activated by death factors or receptors.Important death factors include TNF-α, Fas ligands, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, and tumor necrosis factor-like cytokine 1A.As mentioned earlier, during IRI, reperfusion cells release TNF-α and activate the c-Jun N-terminal kinase (JNK) pathway to stimulate the production of ROS.The accompanying oxidative stress will further stabilize the phosphorylation of JNK and accelerate cell death. 110If TNF-α persistent increase will induce the TNFR-related death domain (TRADD) to combine with it to synthesize TNF α-TRADD.TNF α-TRADD and Fas FADD can induce and activate caspase 8 and 10, then enzymolysis activates downstream caspase 3, 6, and 10, and then starts the cell apoptosis. 111However, cell apoptosis in IRI is not as typical as the necrosis mentioned below.
Necrotizing apoptosis is also programmed cell death, but its impact on organisms completely differs from previous cell apoptosis.The main characteristics of necrotic apoptosis are cell swelling, organelle disintegration, and leakage of intracellular components, which often cause severe inflammatory reactions in ischemic tissues. 112,113As a regulatory mode of death, the main factors triggering necrotic apoptosis are the interacting serine threoninekinase 3 (RIPK3) and mixed lineage kinase-like domain (MLKL). 114RIP3 can enable TNF-α driven cell death changes from apoptosis to necrotic apoptosis.When Caspase-8 is depleted or the inhibitor of apoptosis protein is deficient, TNF receptor I will promote necrotic apoptosis. 113The assembly of the necrotic complex is mainly related to RIPK1/RIPK3 interaction and MLKL activation.RIPK3 induces phosphorylation of MLKL, leading to oligomerization and translocation of MLKL to the lobules within the plasma membrane, ultimately increasing plasma membrane permeability and cell death. 114OS-induced DNA damage also promotes the formation of necrotic complexes by activating poly (ADP-ribose) polymerase (PARP), further accelerating cell death.Due to the close relationship between necrotic apoptosis and the occurrence of inflammation in the human body, understanding the molecular mechanism and pathophysiological significance of necrotic apoptosis remains an essential goal of therapeutic IRI research.
The role of autophagy in IRI is bidirectional, which can both protect cells and disrupt them.Appropriate mitochondrial autophagy can clear partially damaged mitochondria during ischemia and reduce subsequent damage. 115During the reperfusion phase, the levels of Ca 2+ and ROS increase in the cells, while oxidative stress inhibits the activity of rapamycin mechanistic target of rapamycin (mTOR), leading to the formation of UNC51like kinase 1 complexes and PI3K III class, which induce autophagy.However, autophagy cannot clear all damaged mitochondria, and when autophagy clearance capacity is exceeded, it can lead to cell death. 116,117

Inflammation
During reperfusion, producing a large amount of ROS activates the nuclear factor-κappa B (NF-κB) gene, further stimulating the secretion of TNF-α by endothelial cells and macrophages. 118Conversely, TNF-α can induce cell apoptosis through a sphingosine-dependent mechanism.
On the other hand, it can also cause leukocyte infiltration in damaged tissues, increase the permeability of vascular endothelial cells, produce a no-reflow phenomenon, and aggravate reperfusion injury. 119,120At the same time, activated cells release many inflammatory factors, such as IL-1, IL-6, IL-8, IL-10, IL-18, and so on. 121

Nephroprotective effects of H 2 S
Renal IRI is a major predisposing factor for the development and progression of acute kidney injury (AKI). 122,123I is a complex clinical syndrome characterized by a rapid decline in renal function, such as decreased glomerular filtration rate (GFR) with increased creatinine and urea nitrogen, water-electrolyte disturbances, acid-base imbalance, oliguria, or even anuria. 124,125AKI is often associated with severe complications, and the high mortality rate places a significant burden on the healthcare system. 1268][129] In addition to antioxidant effects, H 2 S may exert renoprotective effects through several other mechanisms.First, H 2 S can induce vascular relaxation by opening K ATP channels in endothelial and renal vascular smooth muscle cells, thereby increasing renal blood flow. 47,130Second, H 2 S can potentially protect renal function by inhibiting Ang II in the renin-angiotensin-aldosterone system. 131In addition, some investigators have also found that AP39, a mitochondrial-targeting H 2 S donor, can reduce ROS levels, protect mitochondrial function, and reduce renal epithelial cell injury.However, this protective effect is dose-dependent.Ischemic liver tissue is extremely susceptible to more severe liver dysfunction and failure after reperfusion occurs. 133,134More seriously, hepatic ischemia-reperfusion (HIR) can also affect the success of liver resection or transplantation and increase the risk of death for the operator. 133,135The risk of death is increased.7][138] Some experiments have shown that the expression levels of endogenous H 2 S and CSE are elevated in the tissues of HIR rats, and the researchers speculate that this may be due to the self-protective response of the organism induced by HIR.1][142][143][144][145] However, it has also been found that endogenous H 2 S may exacerbate HIR-induced liver injury in the context of insulin resistance, so H 2 S should be used with caution in this situation. 1461.7Retinal protective effect of H 2 S IRI to the retina is the cause of many retinal vascular diseases, such as diabetic retinopathy (DR), glaucoma, retinal artery occlusion, and so on.147 It is mainly caused by generating and accumulating large amounts of ROS during ischemia and reperfusion.It causes a series of oxidative stress and inflammatory responses that promote irreversible damage to retinal ganglion cells, which may eventually lead to vision loss or even blindness.148 In a study more than a decade ago, researchers injected an H 2 S donor (ACS67) into the vitreous humor of rats with retinal IRI.Subsequently, they found that ACS67 could regulate GSH levels and inhibit apoptosis of retinal ganglion cell-5 (RGC-5) cells induced by oxidative stress, thus exerting a protective effect.149 Another experiment found that direct inhalation of H 2 S for pretreatment prior to retinal IRI in rats reduced the mortality of RGC. 150 In a 2016 study, it was first proposed that enzymes involved in the generation of H 2 S and related pathways are activated during retinal IRI and may have the ability to induce retinal neovascularization.151 In addition, H 2 S may also protect retinal ganglion cells by inhibiting the production of inflammatory factors and activating signaling pathways involved in mediating the protection of mitochondrial function and diastolic vascularity.[152][153][154] 4.1.8 Tesicular protective effect of H 2 S Testicular torsion is a urological emergency that occurs in children and requires immediate surgical treatment; however, despite successful surgical intervention, the incidence of associated complications (such as testicular atrophy and infertility) ranges from 40 to 60%. 155,156Postoperative IRI is the main cause of testicular damage, and previous studies have demonstrated that testicular IRI is closely related to excessive production of ROS, with subsequent massive production of inflammatory factors, oxidative stress, and apoptosis further exacerbating tissue damage. 157,158The subsequent high production of inflammatory factors, oxidative stress, and apoptosis further aggravate the tissue damage.In the last 2 years, studies have revealed that H 2 S may have potential therapeutic effects in protecting testicular tissue. 159,160Bozkurt et al. 160 first investigated the role of H 2 S in IRI in testicular torsion.They found that H 2 S administration inhibited oxidative stress and suppressed the expression of TNF-α, APAF-1, and iNOS to reduce tissue damage. 160Myeloperoxidase (MPO), malonaldehyde, and advanced oxidation protein products (AOPP) are markers of lipid peroxidation, and Yuksel et al. 159 found that NaHS could effectively reduce the expression levels of MPO and AOPP.Meanwhile, Johnson scores were significantly higher in the H 2 S administration group, suggesting that H 2 S can improve spermatogenic function in IRI testes. 159However, there are still relatively few related studies.The mechanism of the protective effect of H 2 S in testicular IRI is still unclear, and we need to conduct more in-depth studies.

H 2 S and organ transplantation
Organ transplantation is the treatment modality of choice for organ failure or severe lesions.][164] Researchers have tried adding additives to the graft preservation solution to discourage cold IRI.Among them, richer results have been achieved in adding H 2 S donors.][171][172] This protective function mainly relies on antioxidant, apoptosis, anti-inflammatory, and vascular mechanisms.So far, in the study of H 2 S donor amelioration of cold IRI, AP39 has shown a high potential to target the mitochondria of donor organs and promote H 2 S entry.Nevertheless, AP39 is an experimental donor and is not clinically licensed, so it is important to find an H 2 S donor that is clinically licensed, which will further the process of moving H 2 S donors from the lab to the clinic.Previously, we mentioned sodium thiosulfate-supplemented (STS) as an antidote that the United States Food and Drug Administration (US FDA) had approved.Some researchers have found that supplementation of STS in the UW solution in which kidneys are stored attenuates tubular necrosis and apoptosis, increases transplant survival, and improves transplant function. 173However, there are fewer reports on the inhibition of cold IRI by STS in SCS, and the specific molecular mechanisms still need to be determined.However, adding STS to the preservation solution is a more economical and convenient method, preventing cold IRI in SCS and improving the grafting success rate.

Anti-inflammatory effects of H 2 S in the cardiovascular system
Inflammation is another hallmark of endothelial dysfunction, and under pathological conditions, transcriptional activation of inflammatory adhesion factors leads to leukocyte and macrophage enrichment of the vascular lining, which induces a range of cardiovascular diseases. 174It has been reported in recent years that H 2 S may be an inflammatory modulator and play an anti-inflammatory role in the cardiovascular system. 175,176In a 2006 study, we reported that blocking endogenous H 2 S synthesis promoted leukocyte adhesion, which the administration of exogenous H 2 S donors conversely inhibited, and that this may be achieved by activating K ATP channels. 84Later reports support the idea that H 2 S has an anti-inflammatory effect.It has been reported that the endogenous H 2 S synthase CSE may be involved in the pathogenesis of atherosclerosis.When specifically absent, CSE enhances monocyte adhesion and accelerates endothelial damage and the atherosclerotic process.It is noteworthy that the use of H 2 S donors had the opposite effect.The underlying mechanism may be related to CSE-H 2 S-induced persulfuration at the Cys-13 position. 177Another study in the same year demonstrated the anti-inflammatory ability of H 2 S from a different perspective, as the investigators found that the H 2 S donors NaHS and GYY4137 increased the expression of mRNA for Sirtuin-1 in arteries and induced its persulfuration, thereby inhibiting arterial inflammation and AS. 178TNF-α has an obvious proinflammatory effect, and it can promote vascular inflammation by inducing endothelial cells to produce adhesion mediators such as monocyte chemotactic protein-1 (MCP-1).It has been found that H 2 S can inhibit the shedding of TNF-α and the release of MCP-1, thereby inhibiting inflammation. 179[182]

Myocardial protective effect of H 2 S
When the blood perfusion and oxygen supply of the myocardium are severely reduced, extensive cell death may occur, leading to myocardial infarction. 183,184As mentioned earlier, although restoring blood supply can alleviate ischemia to a certain extent, it can also lead to a series of more serious reactions, such as oxidative stress, cell damage, and even death.6][187] We found that H 2 S may have a protective effect on myocardial cells through the following five mechanisms.Massively production of ROS during reperfusion is the main cause of oxidative stress responses.However, H 2 S can inhibit its production by regulating ROS signaling pathways, such as inhibiting NF-κB and JAK2-STAT3 pathways to reduce ROS levels. 188At the same time, H 2 S can also increase the expression levels of superoxide dismutase (SOD) and GSH in IRI tissues, both of which are antioxidant enzymes that protect cardiomyocytes by converting peroxides (H 2 O 2 ). 16,189In addition, H 2 S can promote nuclear translocation of nuclear-factor-E2-related factor-2 (Nrf2), an important antioxidant transcription factor that increases transcription of antioxidant proteins and reduces apoptosis and mitochondrial damage. 190n the process of apoptosis, Bcl-2 plays a crucial role.It has been reported that H 2 S is able to reduce the proportion of apoptotic cells in the myocardium of IRI mice by upregulating the expression level of Bcl-2 and decreasing the expression of Bax and cysteine 3. 191 Apoptotic proteins (IAPs) can block the apoptotic cascade response, and it has been found that H 2 S can inhibit apoptosis by affecting the phosphorylation of IAPs and cysteine aspartase recruitment structural domains.
H 2 S protects cardiomyocytes through a bidirectional action in autophagy. 192Tissue or cellular ischemia leads to the development of cellular autophagy, and moderate cellular autophagy facilitates the repair of damaged cells.It has been demonstrated that H 2 S can exert cytoprotective effects by promoting autophagy, which is associated with NLRP3 inflammatory vesicles.Excessive autophagy exacerbates cellular IRI, and H 2 S can activate the PI3K/serum glucocorticoid-regulated kinase 1 (SGK1)/glycogen kinase synthase 3β (GSK3β) signaling pathway and PI3K/Akt/mTOR signaling pathway to inhibit autophagy and provide protection for IRI cardiomyocytes. 193,194n the current study, H 2 S can inhibit the inflammatory response of cardiomyocytes, which is one of the important mechanisms for its cardioprotective effect. 195,196In earlier years, some researchers found that certain H 2 S donors can reduce leukocyte adhesion and infiltration, and this effect seems to be related to the activation of K ATP channels. 84,197n addition, administration of H 2 S treatment before the ischemic tissue regains blood supply also prevents the activation of NF-κB and reduces the production of proinflammatory mediators, with the most significant reduction of IL-1 and IL-6. 198,199Increased expression levels of TNF-α during reperfusion promote interaction between leukocytes and endothelial cells, resulting in increased infiltration of inflammatory cells in the IRI region of the myocardium, which leads to more severe myocardial injury, so inhibition of TNF-α expression may attenuate myocardial injury.It has been found that H 2 S can inhibit the adhesion of inflammatory cells and release associated inflammatory factors caused by TNF-α activation, significantly reducing the expression levels of MCP-1, adhesion factors, and so on. 179he role of mitochondria is particularly important in mammalian growth and development, providing energy for the basic metabolism of the body.When myocardial IRI occurs, the function of mitochondria is severely impaired, leading to further myocardial damage.It has been found that NaHS can reduce mitochondrial malondialdehyde levels in ischemic cardiomyocytes while elevating the activities of SOD and glutathione peroxidase, allowing the preservation of mitochondrial function. 200In addition, H 2 S also increased the efficiency of complexes I and II of the oxidative respiratory chain in mitochondria.It inhibited cytochrome oxidase, reducing the metabolism of cardiomyocytes to a preconditioned state, thereby reducing cardiomyocyte damage. 201,2023 H 2 S and the nervous system

H 2 S and neurodegenerative diseases
H 2 S plays an important role in the CNS.In early times, H 2 S was recognized as a neuromodulator that played a role in enhancing long-term memory in the hippocampal region of the brain in particular. 6Several studies have found that abnormal expression levels of endogenous H 2 S and the transsulfuration pathway may be associated with neurodegenerative diseases.ND, which includes Alzheimer's disease (AD), Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, and spinal cerebellar ataxia, is characterized by a pathological condition in which proteins are misfolded and aggregated, resulting in neuronal damage or even loss, which can in turn lead to cognitive and motor dysfunction. 203,204We mentioned above that CSE and 3-MST are predominantly found in neurons, and several studies have reported reduced or absent CSE expression in different NDs, with or without reduced protein persulfate translation. 86,205,206Oversulfuration modification may be a potential redox switch, and a growing number of studies suggest that H 2 S-mediated oversulfuration modification may be involved in regulating oxidative stress, apoptosis, autophagy, and other processes to maintain normal physiological functions of cells. 16,207he current study found that appropriate concentrations of H 2 S exert neuroprotective effects in ND, and in AD, phosphorylated Tau protein aggregates and misfolded β-amyloid are its main features. 208H 2 S has been shown to cause peroxidative modification of GSK3β and inhibit phosphorylation of Tau proteins.At the same time, H 2 S has been shown to inhibit the expression of amyloid precursor protein cleaving enzyme 1 (BACE1), which, in turn, reduces β-amyloid activity and prevents the development of AD. 206,209 In Parkinson's disease, H 2 S has been found to sulfurize the Parkin protein to promote E3 ligase expression and also induces peroxisulfuric modification of SIRT1 to increase mitochondrial activity, reduce neuronal damage, and further exert neuroprotective effects on neuronal cells. 205,210H 2 S-induced S-sulfonylation of Nrf2 in Huntington's disease inhibits oxidative stress, thereby promoting autophagy of misfolded proteins and suppressing Huntington's disease progression. 211In summary, H 2 S has the role of a neuromodulatory transmitter.It can exert anti-inflammatory, antiapoptotic, and antioxidative stress effects in neuronal cells, so it can be used as a potential neuroprotective agent in neurodegenerative diseases.However, high concentrations of H 2 S accelerate the progression of ND and aggravate neuronal cell damage.Therefore, our attitude regarding the research on using H 2 S as a neuroprotective agent should be cautious and in-depth.

4.3.2
Neuroprotective effects of H 2 S IRI of the brain is an important cause of ischemic stroke, mainly manifested by necrosis or softening of ischemic brain tissue and focal neuronal damage. 212,213Stroke is the most common cause of disability in developed countries, and its high morbidity and mortality pose a great threat to the health of the whole population 214,215 Therefore, researchers need to find ways to detect and prevent ischemic strokes early.Fortunately, it has been found that appropriate concentrations of H 2 S have a neuroprotective effect on IRI in brain tissue. 216,2179][220][221] The detailed mechanism is shown in Figure 5.In addition, it has been reported that H 2 S can also reduce infarct size and restore neurological function by modulating the expression of NMDA receptor expression levels, thereby activating the CREB pathway and improving neuronal cell survival. 222,223owever, another study showed that high concentrations of H 2 S inhibited COX activity in experimental mice, leading to brainstem toxicity and respiratory depression. 224hese suggest that H 2 S plays a dual biological role in the brain. 225,226Although the potential mechanisms of H 2 S in neuroprotection are poorly well understood and refined, there is no doubt that as a multitargeted neuromodulator, H 2 S has a very bright application in treating ischemic stroke.

Nonalcoholic fatty liver disease
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, which is mainly related to the abnormal accumulation of triglycerides in hepatocytes. 227CSE, CBS, and 3-MST are all expressed in the liver, which contribute to the endogenous production of H 2 S in the liver, and the disruption of the metabolism of H 2 S may be related to the development of liver disease.In mice given a highfat diet (HFD), researchers found that the HFD induced lipid accumulation in the liver and disrupted normal liver structure compared with the normal diet group.However, the H 2 S-treated group reversed this phenomenon, improving liver structure, reducing triglyceride and total cholesterol accumulation, decreasing fatty acid synthase expression, and increasing SOD and glutathione peroxidase activity.This experiment showed that H 2 S alleviated fatty liver by improving lipid metabolism and increasing antioxidant capacity. 228It has also been shown that plasma H 2 S levels are positively correlated with HDL cholesterol and negatively correlated with LDL cholesterol. 229Regarding the mechanism by which H 2 S affects NAFLD, it is still unclear.Still, a previous study by our group found that H 2 S reduced apoptosis and promoted autophagy through the ROS-mediated PI3K/AKT/ mTOR signaling pathway, thereby improving HFD-induced NAFLD. 230

H 2 S and IBD
IBD is a chronic inflammatory condition of the gastrointestinal tract that includes two main types: ulcerative colitis and Crohn's disease. 231Inflammatory response and oxidative stress are the main features of IBD. 232When inflammation occurs, neutrophils infiltrate in large numbers, generating ROS, inducing the formation of neutrophil extracellular traps (NETs), and initiating chromatin polymerization. 233,234It has been found that H 2 S donors can induce neutrophil apoptosis and prevent the formation of colitis NETs, thus exerting an anti-inflammatory effect. 235In addition, another study found that H 2 S donor treatment reduced the expression level of the oxidative stress marker 3-NT and increased the expression of the antioxidants GSSH and SOD in a chemically induced IBD model.This suggests that H 2 S may play a cytoprotective role in reducing oxidative stress in IBD by inducing the production of antioxidants. 236In colitis, H 2 S reduces neutrophil infiltration and decreases the expression of several proinflammatory factors, such as IL-6, IL-1β, TNF-α, and NO. 237,238In addition to acting alone, H 2 S can also act as an anti-inflammatory in conjunction with NO or CO. 8,239 Although most studies have demonstrated that H 2 S exerts an anti-inflammatory effect, some experiments have found that higher concentrations of H 2 S increase cytotoxic effects and exacerbate the effects of IBD. 64,240Therefore, the relationship between H 2 S and IBD is complicated, and the effect on IBD is related to the concentration of H 2 S, the type of donor, and the release rate, for which we should carry out a more in-depth and accurate study.

H 2 S and cancer
Cellular metabolic reprogramming is one of the hallmarks of cancer and an inevitable requirement for the survival and proliferation of cancer cells, which helps them to adapt more rapidly to their environment and gain energy. 241In recent years, protein S-persulfidation (P-SSH) has gradually entered the field of vision of researchers, expanding a new way of thinking in cancer research.H 2 S is the most widely studied sulfur-containing gaseous transmitter, and its role in cancer is known to be a double-edged sword, with low concentrations of H 2 S promoting the development of cancer and high concentrations having the opposite effect. 242So there are two options for using H 2 S to treat cancer, that is, inhibition of endogenous H 2 S synthesis or exogenous donor supplementation of H 2 S. 19 Currently, the molecular mechanisms of H 2 S in cancer are uncertain, but one more recognized mechanisms is the persulfidation modification of cysteine residues. 243A deeper understanding of P-SSH's mechanism can help us better develop strategies to inhibit endogenous H 2 S and thus treat cancer.Overcoming oxidative stress is particularly important for the development of cancer cells.Cysteine is a precursor for the synthesis of GSH and is also involved in the antioxidant response.Therefore, ensuring sufficient cysteine is also important for the development of cancer. 244,245P-SSH protects Cys residues from oxidative damage and reduces them to natural thiols under certain conditions, thus preserving protein function. 246In basal-like breast cancer (BLCL), CBS overexpression has been reported to increase P-SSH, which shields BLBC cells from damage caused by oxidative stress and promotes tumor cell proliferation and migration. 247lterations in signaling pathways are also one of the characteristics of cancer, and it has been found that several key molecules of signaling pathways are already sulfurmodified in different cancer types.For example, PTEN, an oncogenic factor of the PI3K/Akt signaling pathway, and protein tyrosine phosphatase 1B (PTPIB). 248,249It has been reported that under the induction of H 2 S, PTEN undergoes P-SSH modification at Cys-71 or Cys-124 and PTPIB at the Cys-215 site.P-SSH of these two molecules would inhibit themselves, activating the PI3K/Akt pathway and enhancing tumor cell proliferation. 248,249][252] Epithelial-mesenchymal transition (EMT) is mandatory for tumors to undergo migration and invasion. 253,254It has been found that silencing the endogenous H 2 S synthase CBS inhibits the EMT process in pancreatic ductal adenocarcinoma cells (PDAC). 255The experimental results showed that silencing CBS inhibited the migration and colony-forming ability of PDAC.Meanwhile, cells silenced with CBS showed decreased expression of WNT5A, SANIL and decreased phosphorylation level of STAT3.STAT3 is an upstream molecule required for the Wnt signaling pathway, so the decrease in phosphorylation may further inhibit the Wnt pathway, thus hindering the EMT process in tumor cells. 256To dig deeper, the researchers did a metabolomic analysis.They found that silencing CBS leads to suppressed levels of protein persulfate, loss of oxidative protection of STAT3, and decreased levels of phosphorylation. 257,258n summary, cancer cells can utilize P-SSH to increase their ability to proliferate and migrate, are less susceptible to oxidative stress, and can better adapt to their environment. 259However, it is currently difficult to distinguish between H 2 S-mediated P-SSH and the organism's RSSH, making further mechanism exploration difficult.In addition to the endogenous H 2 S blocking therapies we mentioned above, providing exogenous H 2 S donors is also an effective option in suppressing cancer.GYY4137 inhibited the proliferation and migration of colorectal and hepatocellular carcinoma, and HA-ADT inhibited the development of breast and esophageal squamous carcinoma. 260,261[264][265][266]

Sulfates
Sulfates are currently the most common H 2 S donors in biological research, such as sodium sulfide and NaHS, and have been shown to have protective effects on cells in disease states in multiple studies. 238Both sodium sulfide and NaHS exhibit a crystalline powder appearance, are easily soluble in water, and can provide H 2 S more directly.In early studies, Zhao et al. used NaHS aqueous solution to release H 2 S and found that it can reduce systemic arterial pressure, indicating that H 2 S has the characteristic of relaxing blood vessels. 47This has been verified in the research of Yoo et al.; in addition, they also found that the reduction of H 2 S donors will lead to the reduction of cardiac output, which will lead to the reduction of systemic arterial pressure. 267This phenomenon does not depend on the regulation of the CNS. 267In multiple studies, H 2 S released by exogenous donor NaHS can protect against organ damage, such as myocardial damage, 268,269 liver, 270 brain, 271 kidney, 269 and so on.However, the chemical properties of sulfide salts are not stable, and the dosage and speed of H 2 S produced after direct dissolution in water are uncontrollable.The release of a large amount of H 2 S can cause a sudden drop in blood pressure, which has adverse effects on experimental animals.

Lawesson reagent and their derivatives
Lawesson reagent (LR) is a common and readily available sulfur ion agent that can be an H 2 S releaser.The molecule of LR contains a quaternary ring with alternating sulfur and phosphorus atoms.Under high-temperature conditions, the sulfur/phosphorus quaternary ring opens to form two unstable RCheicalbook-PS2 (R-PS2), which decompose and release H 2 S. 11,272 Compared with sulfide salts, LR releases H 2 S more slowly. 11However, LR's detailed release molecular dynamics still need to be clarified, and it lacks water solubility, so it has not been widely used as an H 2 S donor.GYY4137 is a new water-soluble H 2 S donor synthesized based on LR reagent, which can slowly release H 2 S. Some studies have found that GYY4137 has the function of dilating blood vessels to resist hypertension. 273ot only that, it can also exert myocardial protection and prevent myocardial ischemia and reperfusion injury by inhibiting inflammation, reducing cell apoptosis, and reducing ROS production. 274,275In addition to its myocardial protective effect, some scholars have found that in IRI, GYY4137 increases antioxidant activity by activating the Nrf2 signaling pathway, which can effectively alleviate renal injury. 276This protective effect has also been reported in testicular torsion and intestinal injury. 277,2780][281] It can also relieve inflammation and reduce intestinal dysfunction when the intestinal barrier is compromised. 282

Sodium thiosulfate-supplemented
Sodium thiosulfate is also a water-soluble H 2 S donor with minimal side effects, and its chemical formula is Na 2 S 2 0 3 .][285] As mentioned earlier, in the metabolism of H 2 S, H 2 S can be oxidized to thiosulfate, and in turn, STS can also become a source of H 2 S. When the body is in a state of hypoxia, H 2 S can be regenerated in thiosulfate through 3-MST and rhodase. 286In addition to releasing H 2 S, STS is also an effective antioxidant that has been proven to have strong protective effects in different organ injuries, such as acute liver injury, 287 acute lung injury, 288 brain injury caused by IRI, 289 myocardial injury, 290 kidney injury, 173 and so on.
Studies have shown that STS may have antiinflammatory effects and protect vascular endothelial cells.H 2 S seems inhibit the NF-κB signaling and exerts anti-inflammatory effects. 291Because of this cytoprotection on IRI, STS therapy has excellent potential in organ transplantation.The organ preservation solution added with STS is expected to become a simple, cheap, and safe new treatment strategy, which can reduce the transplant sequelae and improve the success rate. 292

5.1.4
Natural H 2 S donors and derivatives Some natural foods can also serve as donors of H 2 S. Garlic is considered a good preventive food and has been found to have great medicinal research value. 293,294Allicin is a metabolic active substance in garlic, which can be decomposed to produce diallyl polysulfides, such as diallyl sulfides, diallyl disulfides, and diallyl trisulfides (DATS). 295oreover, these sulfides can react with GSH to produce H 2 S. 296 However, due to the rapid release of H 2 S in water by DATS, some laboratories have utilized exploiting mesoporous silica nanoparticles (MSN) as a carrier to construct a new H 2 S release system (DATS-MSN). 189DATS-MSN can release H 2 S more slowly and controllably.In this study, compared with traditional H 2 S donors (NaHS, DATS, and GYY4137), DATS-MSN showed better cardioprotective effects. 189

AP39
In addition, scientists have synthesized an H 2 S donor targeting mitochondria (AP39).AP39 can reduce intracellular oxidative stress and proinflammatory factor gene expression, maintain cell vitality, ensure mitochondrial energy and DNA integrity, and play an anti-inflammatory and antioxidant cytoprotection. 297In mouse heart transplantation experiments, studies have found that adding AP39 to an organ preservation solution can significantly improve cell viability and reduce cold IRI and tissue fibrosis. 169AP39 can significantly reduce ROS production in mouse pancreatic transplantation experiments and improve pancreatic island function. 172These studies undoubtedly demonstrate the significant potential of AP39 in preventing and treating IRI in organ transplantation.
As an H 2 S donor, in addition to protecting cells, AP39 can induce vascular relaxation by stimulating NO signaling and activating K ATP channels. 298The development of AP39 shows that the development of specific target donors of H 2 S in subcellular organelles has great potential in future biological research.

Potential and future directions of H 2 S therapy
0][301][302][303][304][305][306][307] For exogenous H 2 S therapy, the manner of H 2 S delivery is important.At present, the main methods of H 2 S delivery include direct inhalation and H 2 S donor.However, all these delivery methods have certain limitations.Gaseous H 2 S has a pungent odor and poor safety, and the concentration is difficult to control and cannot be delivered in a targeted manner.Hence, data stability could be improved and applied to human beings. 308norganic sulfates are currently the main H 2 S donors in biology and clinical trials, but they are still poorly targeted and require large amounts to be administered, which can lead to adverse reactions. 309Although recent studies have identified many small molecule H 2 S donors, they still perform poorly regarding stability, solubility, targeting, and half-life. 309Therefore, designing safe, controllable, and stable targeting methods for H 2 S delivery is particularly important.Nowadays, the main way to constitute an H 2 S delivery system is to combine H 2 S donors with polymers by covalent linkage and physical entrapment.Most of these polymers, such as micelles, 310,311 hydrogels, 312 liposomes, 313 and nanoparticles, have good biocompatibility.The delivery systems optimize the therapeutic efficacy with higher stability and bioavailability compared with small molecule H 2 S donors.
To further improve the efficacy of H 2 S therapy, H 2 S delivery systems with intelligent properties have been carried out in recent years.Intelligent delivery systems allow for a stable and controlled release of H 2 S by specifically targeting and responding to the pathological microenvironment and external stimuli.In addition, the system monitors the release of H 2 S through the use of materials with imaging capability. 308Zhao et al. 314,315 designed an N-mercapto (N-SH)-based H 2 S delivery system that not only releases H 2 S in a controlled manner, but also displays potent myocardial protection in a mouse model of myocardial IRI.Takatani-Nakase et al. 316 found that ADT nano micelle could release H 2 S in an in vitro ischemia model and were more effective in blocking cardiomyocyte apoptosis than NaHS and ADT-OH.Sun et al 317 .constructed a targetable H 2 S delivery system (DATS@MION-PEG-LF) based on mesoporous iron oxide nanoparticles (MION).The system is mainly composed of MION, DATS, polyethylene glycol (PEG), and lactoferrin (LF).To prolong circulation time, the researchers modified PEG into MIONs, while LF helps the system target the brain through the blood-brain barrier.The study found that DATS@MION-PEG-LF can be taken up by neuronal cells and cardiomyocytes, protecting the brain and heart by exerting antiapoptotic and oxidative effects in IRI.More notably, this process can also be visualized by MRI. 317Hsieh et al. prepared a nanoparticle system loaded with DATS (DATS@MPs). 318Compared with free DATS, DATS@MPs can release H 2 S more slowly and for a more extended period.In a mouse model of hind limb ischemia, DATS@MPs can limit apoptosis, protect cells, and promote angiogenesis, which is beneficial for hind limb recovery. 318][321][322][323] He et al. used bovine serum albumin (BSA) as a template to design MnS@BSA.MnS@BSA dissociates in a weakly acidic environment, releasing H 2 S and Mn 2+ .][327][328] We summarize the progress of the H 2 S smart delivery system in preclinical studies (summarized in Table 1) with the aim of more clearly and unambiguously describing the therapeutic potential of H 2 S and donors.
However, smart H 2 S delivery systems are still in their infancy, and there is still a long way to go from the lab to the clinic.First, the pharmacological research of H 2 S needs to be more profound, and the detailed molecular mechanism and targets of its action in vivo need to be clar-ified.Second, it is not easy to monitor the location and concentration of H 2 S accurately using current commonly used technologies.Therefore, H 2 S delivery systems rely on more in-depth biological research.However, we believe that through innovation and improvement in all aspects, the intelligent H 2 S delivery system will eventually unleash its great potential.

DISCUSSION
There is increasing evidence that reasonable concentrations of H 2 S can have protective effects in physiological and pathological conditions, possibly through antiapoptosis, regulation of autophagy, and inhibition of oxidative stress and inflammation.The growing understanding of the important biological effects of H 2 S, such as vasodilatory, cytoprotective antioxidant, and anti-inflammatory effects, as well as its signaling pathway mechanisms, has facilitated the translation of the highly promising cytoprotective functions of H 2 S into more viable clinical therapeutic modalities.Key to this is the effective design of H 2 S donors to deliver the desired therapeutic effects.As discussed earlier, designing stable, controlled H 2 S donors that maintain a stable and slow release of H 2 S over time is preferable for clinical applications, and much of the physiological utility of H 2 S is derived from its redox properties.The uncontrolled and rapid release of H 2 S donors rapidly alters the redox state of cells, which has a much greater impact on cells than its beneficial physiological functions.With rapidly increasing H 2 S concentrations, the distribution of each different oxidation state sulfide is vastly different from the normal physiological state, yet each sulfide has its unique physiological properties.
The volatility of H 2 S and its rapid metabolism makes the development of H 2 S donors uniquely challenging compared with the development of other small molecule donors, which are highly volatile and are always in a dynamic, volatile-soluble equilibrium.In addition, many of the current H 2 S donors are polysulfides, both the donor itself and the by-products of H 2 S fraction production, so it is often difficult to distinguish whether the physiological effects of such donors are derived from H 2 S or other polysulfides.Another difficulty in H 2 S research is how to quantify the range of endogenous H 2 S concentrations during human circulation and the changes in H 2 S concentrations during treatment.This is mainly due to the reactive chemical nature of H 2 S and the complex environment of sulfides in vivo.The inability to accurately monitor H 2 S concentrations in the circulatory system or target organs will make it difficult to assess the exact relationship between H 2 S and physiological effects.Therefore, In conclusion, although sulfide generators are not new drugs to date, there is precedent for reducing metabolism, thus providing protection against IRI in humans.For example, hypothermia therapy has been shown to be beneficial for outcomes in a variety of situations, including out-of-hospital cardiac arrest and during myocardial revascularization.Although many issues still need to be addressed, these critical issues must be resolved to move into clinical treatment.However, future multidisciplinary collaborations involving nanomaterials, chemistry, pharmaceutical, and biological disciplines may finally offer a possibility for H 2 S therapy, and we forward to seeing more exciting studies in this area.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no conflict of interest.

R E F E R E N C E S
Figure 4 provides a detailed description of ROS-induced cell death mechanism.

F I G U R E 5
Mechanisms of neuroprotection exerted by H 2 S. H 2 S can reduce the infarct size of cerebellar tissue and restore neurological function through mechanisms such as antioxidant, anti-inflammatory, antiapoptotic, regulating autophagy, protecting mitochondrial function, and vasodilation and generation.By Figdraw.COX-2, cytochrome oxidase subunit 2; γ-GCS, γ-glutamyl cysteine synthetase; iNOS, inducible nitric oxide synthase; mPTP, mitochondrial permeability transition pore; NF-κB, nuclear factor-kappa B; VEGF, vascular endothelial growth factor.

TA B L E 1 235 CAP
Summary of advances in H 2 S donors and smart delivery systems in preclinical studies.model(rat) Decrease of ROS level via downregulation of NF-κB and JAK2/STAT3 pathways 188 NaHS Infarction model(mice) Upregulation of Bcl-2, demoted expression of Bax, IL-1β, and Caspase -PEG-LF Hypoxia/reoxygenation model and CA/CPR model(mice) Antioxidant, anti-inflammatory and antiapoptotic 317 Antiatherogenic NaHS Apolipoprotein-E K.O.model (mice) Inhibition of ICAM-1 and TNF-α signaling pathway 330stroke NaHS Cerebral I/R model(rat) Improved SOD activity, removed oxygen free radicals, inhibited lipid peroxidation, and downregulated the expression of HSP70 219 Relief of NAFLD GYY4137 NAFLD model(mice) Inhibition of hepatic ER stress through the SIRT1/FoxO1/PCSK9 pathway 335 Relief of IBD Lawesson's reagent and SASP Colitis model(rat) Inhibition of NETs formation to exert anti-inflammatory effects Huh7 cell, xenograft model(mice) Inhibition of tumor growth it is important to develop methods to quantitatively detect H 2 S concentrations in vivo for H 2 S research.
Dong-Dong Wu, Zhi-Guang Ren, and Xin-Ying Ji: conceived and supervised the study.Yu-Qing Jin, Hang Yuan, Ya-Fang Liu, Yi-Wen Zhu, Yan Wang, Xiao-Yi Liang, and Wei Gao drafted the manuscript and prepared the figures.All authors read and approved the final manuscript.A C K N O W L E D G M E N T SThis work was supported by grants from the National Natural Science Foundation of China (No. 81802718), the Training Program for Young Backbone Teachers of Institutions of Higher Learning in Henan Province, China (No. 2020GGJS038), the Natural Science Foundation of Education Department of Henan Province, China (Nos.21A310003, 24B310001), and the Foundation of Science & Technology Department of Henan Province, China (Nos.222102310490, 222102310495, 232102310507).